While automated data acquisition methods have enabled the delineation of regulatory pathways, the detailed molecular mechanisms that drive and coordinate these processes remain unknown or incompletely characterized. The overall goal of this proposal is to deduce, at the molecular level, the mechanisms that control an entire signaling pathway, that of the nitrogen regulatory circuit in the model Gram-positive bacterium B. subtilis. In B. subtilis, the metabolism and assimilation of nitrogen is controlled by an unusua network of proteins, distinct from that used by Gram-negative bacteria, that include the central enzyme of nitrogen metabolism, glutamine synthetase (GS), the global transcription regulators, TnrA and GlnR, and the ammonium transport regulator, GlnK. GS synthesizes glutamine (Q), which is the preferred nitrogen source in B. subtilis, while GInR and TnrA regulate the transcription of all protein-encoding genes involved in nitrogen metabolism. In B. subtilis, GS plays a central role not only in nitrogen assimilation, but also transcription regulation by interacting directly with TnrA and GlnR in its glutamine feedback-inhibited form (GS-Q). During nitrogen excess, GS-Q is formed and binds TnrA to prevent the DNA binding activity of this global activator, while it activates the repressor activity of GlnR by an unknown chaperoning mechanism. During nitrogen poor conditions, GlnK interacts with TnrA to facilitate its ability to bind DNA. Thus, the B. subtilis nitrogen assimilation pathway is highly interconnected, ultimately allowing B. subtilis to detect intracellular nitrogen levels and transmit this signal to effect enzme activity and gene regulation. To date, we have determined the enzymatic mechanism of B. subtilis GS at the atomic level, revealing that its catalytic activity and regulation are distinct among GS proteins. The goals of this project are to perform biochemical, structural and in vivo studies to dissect the regulatory mechanisms that control this nitrogen assimilation pathway. The two, multi-part Specific Aims are as follows: (1) Deduce the molecular mechanisms controlling the GlnR regulatory network including GlnR DNA-binding, its regulation by autoinhibition, and the unique chaperone function of GS. (2) Elucidate the TnrA DNA binding mechanism and its activation by GlnK and inhibition by GS-Q. Notably, GS is an established antibacterial drug target. Thus, the detailed structural information obtained in this proposal will provide insight into improved drug development as well as provide new targets, such as TnrA and GlnR, for the design of highly specific, antibacterial chemotherapeutics.